The use of enzymes in industrial applications, and for various biological and medical purposes is well-known. Such materials find use e.g. in the construction of membranes for enzyme therapy, as in artificial kidneys and other artificial organs. They are also used in conjunction with electrodes and related detection devices for medical diagnostics, and as catalysts in enzyme reactors, etc. These and other enzyme uses have been growing rapidly as the availability thereof increases and the unique catalytic characteristics of these materials are better understood.
In general, the limited availability of the various enzymes and attendant high costs have been a serious detriment to their wider use in commercial applications. The use of free enzymes in aqueous compositions is simply not economically feasible because of the excessive loss of the enzyme that results and the difficulty in controlling the amount, activity and stability thereof in such an environment.
As is well known, one of the most advantageous techniques that has been found for making feasible the practical use of enzymes, involves immobilization of enzymes on substrates that serve to confer stability to the enzymes. In such cases, the enzyme is absorbed or absorbed on a substrate in such a manner that a sufficient amount of the enzyme is available for the projected use, and the activity, generally catalytic activity, is not unduly limited by the immobilization mechanism.
Previous approaches to the immobilization of enzymes have employed coupling agents to covalently link the enzyme to the support surface. Typical functionalized supports have included porous and non-porous glass, nickel oxide, silicaalumina, polyacrylamide, polystyrene, albumin and cellulose. Other approaches have physically incorporated the enzyme into porous matrices such as cellophane, controlled pore titania, collagen, silicone elastomers and polyacrylamide gel. The specific activity and stability of an enzyme immobilized by covalent attachment or occlusion depends strongly on the nature of the support and on a variety of operational factors which generally influence the performance of immobilized enzymes, such as the pH and/or temperature stability profile of the bound enzyme.
It has also been recognized for a number of years that swelling smectite clay minerals such as sodium-montmorillonite and hectorite, bind enzymes by an ionic intercalation mechanism, often with retention of catalytic activity. Enzyme immobilization on sodium exchanged or unmodified layered clays has been found to be extremely pH dependent. Below the enzyme isoelectric point, strong interactions for enzyme immobilization are provided. However, above the isoelectric point, where the enzyme become negatively charged, the enzymes are readily desorbed from the clay surface. This behavior is undesirable because the pH for optimum enzyme activity is often above the isoelectric point. Further, enzymes bound to unmodified clays exhibit substantially reduced activity relative to homogeneous solutions and are readily denatured.
More recent studies of alternate routes to enzyme immobilization have recognized a potential significance of hydrophobic binding mechanisms for influencing the efficiency of immobilized enzymes. Such studies, however, have generally been limited in scope and the effect of factors that may relate to such mechanisms are not known.
Organic-clay complexes, which are the reaction products of smectite-type clay minerals as well as certain synthetic material resembling them, are well known and have been widely used as gelling and viscosity control agents for organic liquids such as lubricating oils, linseed oil, toluene and the like. A large variety of highly useful products, such as lubricating greases are produced through use of such gelling agents. The procedures and chemical reactions pursuant to which these organoclays are prepared are also well known.
Among the prior art patents which discuss at length aspects of the preparation and properties of organicclay complexes (organoclays) are U.S. Pat. Nos. 2,531,396; 2,531,427; 2,531,440; 2,966,506; 3,227,657; 3,298,849; 3,422,185; 3,537,994; 3,974,125; and 4,081,496, and U.K. Pat. No. 920,797. Reference may also be made to applicable portions of the standard reference by Ralph E. Grim, "Clay Minerology", 2d Edition, 1968, McGraw Hill Book Company. None of these references nor other disclosures in the prior art have been addressed to or have suggested providing an immobilized enzyme composition or of any means for immobilizing enzymes.
It is evident that there are limitations in the mechanisms that are known for immobilizing enzymes including pH dependence, ready denaturation, inhibited catalytic activity, excessive cost of materials and/or complexity of synthesizing techniques. It would, therefore, be highly desirable to prepare immobilized enzymes which were catalytically active over the broadest possible pH and temperature ranges and would lend itself to the least complex synthetic methodology.
It is, therefore, a primary object of this invention to provide a method for immobilizing enzymes by mechanisms which are substantially pH-independent.
Another object of this invention is to provide new and improved immobilized enzyme compositions.
Still another object of this invention is to provide a method for the pH-independent immobilization of enzymes which is simple to carry out and which employs readily available binding materials for said enzymes.
Yet another object of this invention is to provide new and improved immobilized enzymes which, depending on the nature of the enzyme and on the nature of the immobilizing component, may completely inhibit the activity of the immobilized enzyme, or may exhibit activity substantially comparable to the activity of the free enzyme in homogeneous solution, or may be modified to some other degree of activity.
Another object of this invention is to provide a method for producing immobilized enzyme-organoclay complexes in which enzyme binding is substantially independent of solution pH.
Still another object of this invention is to provide new and improved enzyme-organoclay complexes in which the enzymes are immobilized and in which the binding is substantially independent of solution pH.
Yet another object of this invention is to provide enzyme-organoclay complexes having loadings of immobilized enzymes up to about 40 percent by weight or even greater.
These and still further objects of the invention will become readily apparent to one skilled in the art from the following detailed description, specific examples and drawings.